Anti-tumor Effect Of A Dual Cancer-specific Oncolytic Adenovirus Ad-VT On Ovarian Cancer

Cancer is one of the main causes of human death.In 2020,19.3 million cases of cancer have been diagnosed worldwide,and 10 million have died from cancer.Among them,the incidence and mortality of ovarian cancer rank the eighth among female cancer incidence and mortality.Ovarian is located in the deep pelvic cavity,early stages are not easy to be found,once found,most of them are in the late stage,the current treatment for ovarian cancer is mainly based on the combination of surgery and chemotherapy,but chemotherapy side effects are large,easy to produce drug resistance.Therefore,the search for more effective treatments is still needed.At present,with the rapid progress and continuous development of molecular biology,cell biology and virology,gene therapy has gradually developed into a new type of treatment for ovarian cancer,among which the new oncolytic adenovirus has fully demonstrated a The advantages of irreplaceable treatment have played the dual important role and function of virus therapy and gene therapy,and it is expected that it will develop into an effective treatment for ovarian cancer as soon as possible.Apoptin is an apoptosis-inducing protein derived from chicken anemia virus(CAV).It has no cytotoxicity in normal cells,but can specifically induce tumor cell apoptosis in tumor cells.The length and viability of human telomerase reverse transcriptase(h TERT)are related to cell senescence and immortalization.Due to the rate-limiting effect of the telomerase reverse transcriptase(h TERT)gene and the tight transcriptional inhibition of the catalytic component,most normal human cells lack telomerase activity.However,h TERT expression and telomerase activation have been observed in up to 90% of human malignant tumors,giving them unlimited proliferation capacity.In previous studies,our group has constructed a human type five adenovirus AdApoptin-h TERTp-E1a(Ad-VT)containing apoptin protein and h TERTp promoter,which has the ability to replicate only in tumor cells and specifically kill a variety of tumor cells.In this study,the Ad-VT and its control viruses Ad-T,Ad-VP3 and Ad-MOCK were used to study the in vivo and in vitro inhibitory effects on human ovarian cancer A2780 and SKOV3 cells.Purposes:The inhibitory effect of Ad-VT on human ovarian cancer A2780 and SKOV3 cells was analyzed in vivo and in vitro.Methods:1.Human ovarian cancer A2780 and SKOV3 cells were transfected with p GL4.51,and human ovarian cancer A2780-Luc and SKOV3-Luc cells stably expressing luciferase gene were screened with G418,and the biological characteristics were analyzed and detected.2.The killing effect of Ad-VT on human ovarian cancer A2780-Luc and SKOV3-Luc cells was analyzed by crystal violet staining and WST-1 detection.3.The main ways of Ad-VT killing human ovarian cancer A2780-Luc and SKOV3-Luc cells were analyzed by Hoechst staining,Annexin V flow analysis,caspase activity detection,reactive oxygen species detection and JC-1 staining.4.The effect of Ad-VT on the metastasis ability of human ovarian cancer A2780-Luc and SKOV3-Luc cells was analyzed by cell scratches and Transwell chamber invasion assays.5.Tumor-bearing models of human ovarian cancer A2780-Luc and SKOV3-Luc cells were established in nude mice,respectively,and the inhibitory effect of Ad-VT on human ovarian cancer A2780-Luc and SKOV3-Luc cells in vivo was analyzed by observing tumor luminescence intensity,tumor size and the survival of mice every week.Result:1.Human ovarian cancer A2780-Luc and SKOV3-Luc cells expressing luciferase gene were successfully constructed,and cell lines with stable expression were screened out,and the constructed cells showed no significant biological differences with the original cells.2.Through crystal violet staining and WST-1 assay,it was shown that Ad-VT had killing effect on human ovarian cancer A2780-Luc and SKOV3-Luc cells,and the killing effect was in a dose-effect and time-effect relationship.3.Hochest staining,Annexin V flow analysis,caspase activity detection,reactive oxygen species detection and JC-1 staining showed that Ad-VT mainly killed human ovarian cancer A2780-Luc and human ovarian cancer SKOV3-Luc cells through endogenous apoptosis pathway.4.Cell scratch assay and Transwell chamber invasion assay showed that Ad-VT had significant inhibitory effect on the migration and invasion of human ovarian cancer A2780-Luc and SKOV3-Luc cells.5.Ad-VT can also significantly inhibit human ovarian cancer A2780-Luc and SKOV3-Luc in vivo and prolong the survival of nude mice by constructing the tumor model of human ovarian cancer A2780-Luc and SKOV3-Luc cells expressing luciferase.Conclusion:In this study,human ovarian cancer A2780-Luc and SKOV3-Luc cells were successfully constructed,and a variety of experiments in vivo and in vitro confirmed that Ad-VT has a significant killing effect on human ovarian cancer A2780-Luc and SKOV3-Luc cells,indicating that Ad-VT has the potential to be a treatment drug for ovarian cancer.